PROJECT SUMMARY The most important determinant of cardiovascular health is a person's age, as the risk of cardiovascular disease (CVD) increases significantly as we grow old. In this project, we will generate a new mutant Lmna mouse, as a model to probe the role of permanently farnesylated prelamin A in driving the CVD of physiological aging. LMNA encodes prelamin A, the precursor of the nuclear scaffold protein lamin A. Normally, prelamin A undergoes farnesylation and subsequent proteolytic cleavage by the protease ZMPTE24 that removes the farnesylated C-terminal portion of the protein. In the premature aging disorder Hutchinson- Gilford Progeria Syndrome (HGPS), an internally deleted (?50aa) form of prelamin A called progerin that remains permanently farnesylated causes disease phenotypes, including CVD. However, HGPS mouse models may not effectively model the CVD of physiological aging in unaffected individuals, because progerin is produced in vanishingly small amounts and the ?50aa deletion may impart novel binding properties to this prelamin A variant. On the other hand, compelling recent studies provide evidence that prelamin A, normally a transiently expressed farnesylated precursor that is rapidly converted to mature lamin A, accumulates in vascular smooth muscle cells of aged, but not young, individuals and in atherosclerotic lesions. Although the observed accumulation of farnesylated prelamin A in the vasculature of old human subjects is intriguing, there is no mouse model to directly assess experimentally the role of this protein in the development of CVD. The existing Zmpste24-/- mouse is not ideal for such studies because this enzyme has another critical cellular function besides prelamin A processing, which may confound analysis. Instead, to test the hypothesis that permanently farnesylated prelamin A promotes accelerated CVD, we propose to generate mice with a LmnaL648R mutation. The L648R amino acid substitution abolishes the ZMPSTE24 cleavage recognition site, leading to the accumulation of permanently farnesylated full-length prelamin A, essentially the same species seen in aging vessels. In Aim 1, we will generate knock-in LmnaL648R mice and analyze them for multiple organismal and cellular phenotypes associated with progeria and physiological aging. In Aim 2, we will characterize the development of vascular pathology in heterozygous and homozygous LmnaL648R mice by performing a longitudinal study of vascular stiffness using non-invasive pulse-wave velocity testing over their lifetimes. We will also perform interventional and ex vivo assessments of vascular mechanics, vasoreactivity, vasomechanics and vascular pathology. This R21 proposal involves development ? generation of a novel mouse model to study the potential role of prelamin A in CVD of aging ? and exploration ? studying these mice for the progression of CVD as they age. The potential for reward is huge, as these studies could pave the way for a paradigm-shifting understanding of CVD, the most common cause of morbidity and mortality for old Americans.